1. Technical Field
The present invention relates to a method for producing a target substance by using a microorganism. More precisely, the present invention describes a method to improve production using a microorganism of final target products, such as L-amino acids, nucleic acids, and so forth.
2. Background Art
Methods are well-known for producing substances such as L-amino acids by fermentation using microorganisms. L-amino acids are used not only as seasonings and in foodstuffs, but also as components of various nutritional mixtures for medical purposes. Furthermore, they are used as additives for animal feed, reagents in the drug manufacturing and chemical industries, and as growth factors for production of L-amino acids such as L-lysine and L-homoserine. Microorganisms that can produce L-amino acids by fermentation include coryneform bacteria, Escherichia bacteria, Bacillus bacteria, Serratia bacteria, and so forth.
In the production of target substances by fermentation, most of the raw materials contain saccharides such as blackstrap molasses. Also in amino acid fermentation or nucleic acid fermentation, the culture is performed using a saccharide as the raw material. Although sugarcane and so forth contain abundant amounts of starch, it is rarely used as a raw material, but can be present as a decomposition product of starch, for example, monosaccharides or disaccharides. Starch decomposes in the presence of a solution of a saccharifying enzyme such as amylase, and polysaccharides are decomposed into relatively low molecular weight saccharides, such as glucose, maltose and isomaltose.
When using a starch hydrolysis solution in fermentation, if the fermentation is terminated when all the glucose is consumed, oligosaccharides such as maltose and isomaltose are not assimilated. Escherichia bacteria do not have the ability to assimilate these oligosaccharides, especially isomaltose, and they cause the Maillard reaction with the products of the fermentation, especially L-amino acids, which poses a problem when purifying the fermentation product.
Moreover, once all the glucose is consumed, the remaining oligosaccharides such as maltose that were not consumed due to the presence and consumption of glucose must be consumed, thus extending the culture time and needlessly increasing the heating expense.
A technique has been disclosed to utilize the maltose produced by starch decomposition, specifically, to use a microorganism in which an interaction is attenuated or eliminated between the IIAGlc protein of the glucose PTS and the MalK protein involved in the non-PTS uptake of maltose, resulting in the microorganism being able to take up glucose and maltose simultaneously (U.S. Pat. No. 7,097,999, European Patent Laid-open No. 1254957). It is known that the suppression of the uptake of other oligosaccharides by glucose is generally observed in E. coli and Salmonella typhimurium. That is, when glucose and other carbon sources such as maltose are present during fermentation, glucose is assimilated first, and then the other carbon sources are assimilated. However, the aforementioned technique enables simultaneous assimilation of glucose and maltose.
Furthermore, an α-amylase can be expressed in a cell surface layer to decompose starch into glucose and maltose (which is an α-1,4-glucan), which are then taken up into cells, which can result in an improved ability to produce amino acids (Tateno, T., et al., Appl. Microbiol. Biotech., 74, 1213-1220 (2007)).
Maltose is known to be taken up into cells by the GlvC protein, also known as the PTS maltose enzyme IICB, encoded by the glvC gene of Bacillus subtilis (B. subtilis). Once in the cell, maltose, present as phosphorylated maltose, is hydrolyzed by GlvA (6-phospho-α-glucosidase) encoded by the glvA gene (Schonert S. et al., J. Bacteriol., 2006, June; 188 (11):3911-22). However, the relationship between the glvAC genes and isomaltose assimilation has not been reported to date.
The enzyme α-glucosidase decomposes maltose, sucrose, and isomaltose into monosaccharides, and is present in B. subtilis. α-glucosidase is encoded by the malL gene (Schonert, S., et al., J. Bacteriol., 180, 2574-2578 (1998)).
The presence of such an enzyme as described above has not been reported for coryneform bacteria and Enterobacteriaceae bacteria, including E. coli. Therefore, it is considered in the art that these bacteria cannot assimilate isomaltose, and whether they can take isomaltose up into cells is unknown. Furthermore, a microorganism that extracellularly secretes an enzyme that decomposes isomaltose has also not been reported.